The goal of this project is to define the molecular mechanisms involved in the replication of mammalian retroviruses and in particular, to understand the factors which influence the regulation and expression of viral genetic information. Studies are being carried out with the murine leukemia virus system. Current interest is focused on the organization of the MuLV pol gene and on correlation of genetic structure with pol-associated enzymatic functions. Molecular clones containing MuLV reverse transcriptase sequences are being expressed in E. coli. The enzyme expressed by one of these clones, pRT250, was previously shown to have normal MuLV polymerase activity, but only barely detectable levels of RNase H activity. The deficit in RNase H activity has now been correlated with the absence of almost half the amino acid residues comprising a C-terminal region with homology to the E. coli. and yeast RNases H. These results support the idea that the catalytic sites for polymerase and RNase H are localized to the N- and C-terminal portions of reverse transcriptase, respectively. Experiments to explore the functional relationship between these domains and to identify functionally significant sequences within the domains are in progress. In other work, translational control of viral gene expression is being investigated. Efforts are focused on the role of tRNA in readthrough and ribosomal frameshift suppression at retroviral gag-pol junctions. Suppression of the in-frame UAG termination codon separating the MuLV gag and pol coding regions has been demonstrated in an in vitro system. Yeast tyrosine amber suppressor tRNA as well as partially purified tRNA fractions, including glutamine tRNA, from MuLV-infected cells stimulate readthrough. Further purification of the mammalian tRNA species with suppressor activity is underway. In studies on ribosomal frameshift suppression, the distribution of isoacceptor tRNAs corresponding to amino acids present at or around the frameshift site is being analyzed. The results show that cells infected with HIV, HTLV-l, and bovine leukemia virus differ from control cells by a dramatic increase in the representation of some of these tRNAs in the hypomodified form.